p38 MAPK-MSK1/2-CREB信号通路关键分子在EMT痛经小鼠宫腔微环境中的响应及少腹逐瘀汤的干预作用

This study which is based on the theory of pathogenic cold and blood stasis block, pathogenesis of endometriosis-dysmenorrhea and combined with previous work, was implemented to establish the theoretical hypothesis that key molecules of p38 MAPK-MSK1/2-CREB signaling pathway played roles of activating simultaneously in mice uterine cavity tissue, and to observe the possible interfering action of Shaofuzhuyu decoction. therefore, this study was designed to explore the expression laws of p38 MAPK-MSK1/2-CREB signaling pathway among uterine cavity by employing methods of immunohistochemistry, qPCR and Western blot detection in mice of endometriosis-dysmenorrhea model, and to observe the inter-relations among p38 MAPK, MSK1/2, CREB and 5-HT, ANKA, NO, PGs, IL-lβ, IL-6, IL-8, TNF-α, following cascade excitation effects, and the effects of Shaofuzhuyu decoction on them. Based on inter-relations analysis, this study was supposed to reveal the the mechanisms of endometriosis-dysmenorrhea and essential reasons of Shaofuzhuyu decoction on it from the roles of key molecules of p38 MAPK-MSK1/2-CREB signaling pathway, and to provide theoretical support for prescriptions in applying Wenjing decoction and developing advantages Chinese medicines when treating Endometriosis-dysmenorrhea.

子宫内膜异位症（EMT）痛经为中医治疗优势病种，其证候多为寒凝血瘀型。本研究基于“寒瘀交阻”致痛理论及与炎性反应和疼痛敏化有关的信号通路，建立“p38 MAPK-MSK1/2-CREB通路关键分子在EMT痛经小鼠宫腔微环境中的响应及少腹逐瘀汤干预可能作用”工作假说，以寒凝血瘀型EMT痛经小鼠为对象，采用免疫组化、qPCR和WB技术检测该信号通路关键分子基因蛋白及其磷酸化在宫腔中的表达规律，同时观察相关疼痛介质5-HT、SP、ANKA、ATP、NO、PGs以及炎性因子IL-lβ、IL-6、IL-8、TNF-α等之间的级联激发效应，并应用少腹逐瘀汤进行干预，通过相关性分析，拟从p38 MAPK-MSK1/2-CREB信号通路关键分子角度揭示少腹逐瘀汤对寒凝血瘀型EMT痛经的干预机制，为该复方临床应用和筛选防治EMT痛经优势中成药关键技术提供理论支撑。

本项目结合子宫内膜异位症（EMT）痛经临床以西北寒冷气候易致妇女出现寒凝血瘀病机特点，提出基于“寒瘀交阻”致痛理论，建立“p38MAPK-MSK1/2-CREB信号通路关键分子在EMT痛经大鼠宫腔微环境中的交互响应及少腹逐瘀汤可能干预作用”工作假说，探讨了该信号途径在“寒瘀交阻致痛”中的响应以及少腹逐瘀汤的干预效应，为该复方临床应用和筛选防治EMT痛经优势中成药关键技术提供理论支撑。.研究结果：1.p38MAPK-MSK1/2-CREB通路在寒凝血瘀型EMT痛经模型大鼠子宫微环境中的动态表达（1）寒凝血瘀型EMT痛经大鼠模型的建立与评估：寒凝血瘀EMT痛经模型大鼠一般生存状态较差，EMT痛经表现明显，组间差异具有统计学意义；EMT痛经组随造模时间的延长，移植物体积逐渐增大。EMT痛经模型组子宫组织[Ca2+]i显著升高。（2）EMT痛经模型组在位内膜p38MAPK、CREB、NGF、BDNF表达水平逐渐升高；p-p38MAPK、p-CREB、NKA、SP表达，IL-lβ、TNF-α、IL-6、IL-8、NO的含量呈现阶段性升高；MSK1/2表达逐渐降低，差异具有统计学意义。提示在寒凝血瘀型EMT痛经模型大鼠宫腔微环境中，疼痛敏化及炎症因子存在随造模时间延长逐渐增多的趋势。.2.少腹逐瘀汤通过p38MAPK-MSK1/2-CREB通路干预寒凝血瘀EMT痛经大鼠的效应及分子机制：EMT痛经模型大鼠给予低、中、高浓度少腹逐瘀汤和阳性对照药物孕三烯酮干预后，各治疗组大鼠宏观证候积分改善，少腹逐瘀汤高剂量组恢复较快；EMT痛经表现改善；异位病灶体积缩小，组间差异均具有统计学意义；子宫组织中[Ca2+]i显著降低，p38MAPK、CREB、NGF、BDNF表达下降；p-p38MAPK、p-CREB、NKA、SP表达，IL-lβ、TNF-α、IL-6、IL-8、NO含量的水平均下降，以少腹逐瘀汤高剂量组明显，差异具有统计学意义，中药高、中剂量组及对照组子宫MSK1/2蛋白及在位子宫内膜组织中MSK1/2 mRNA表达明显升高。提示在寒凝血瘀型EMT痛经大鼠的宫腔微环境中，少腹逐瘀汤通过改变p38MAPK-MSK1/2-CREB通路中的炎症因子、疼痛敏化因子基因、蛋白及其磷酸化水平，改善了寒凝血瘀型EMT痛经疼痛，发挥了抑制宫腔微环境炎性状态和疼痛敏化进展的作用。